The present invention relates to a method for inactivating contaminants in blood products, especially whole blood, plasma, fluids comprising cellular blood compounds, and blood derivatives such as clotting factors (factor VIII, factor IX, factor VII, factor II, Factor X, von Willebrand""s factor and the like), fibrinogen, fibronectin, immunoglobulins, albumin, thrombin, anti-thrombin III, plasminogen, and the like, including nonnatural products obtained by biological engineering, as well as the apparatus for carrying out the said method.
The present invention also relates to blood products treated by the method of the invention as well as pharmaceutical and/or cosmetic compositions comprising the said blood products.
The availability of blood products requires, for their use for therapeutic or nontherapeutic purposes, purification techniques which make it possible to obtain products of high purity, and preferably free of contaminants, in particular of viral contaminants.
In blood products, the viral contaminants may be enveloped viruses (HIV, hepatitis B, C, D, E and G viruses, and the like) or nonenveloped viruses (hepatitis A virus, parvovirus, and the like).
For many years, various international or national bodies have introduced increasingly strict standards for the preparation of blood products so as to prevent their application for therapeutic or nontherapeutic purposes when they contain viral contaminants (Council Directives 65/65 EEC, 75/319/EEC and 89/381 EEC).
At the European level, the CPMP standards (CPMP/BWT 268/95 and CPMP/BWP/269/95 rev 2) require the use of certain treatments against enveloped or nonenveloped viruses.
It is in particular mentioned in these documents that an inactivation step using heat (dry or steam) or using pasteurization in the preparation of clotting factors is effective against the hepatitis A virus, but would not be very effective against other nonenveloped viruses, in particular parvoviruses.
On the other hand, a treatment comprising a chemical inactivation step (by addition of solvents-detergents) is effective for enveloped viruses but ineffective for treating nonenveloped viruses.
It is also known to use certain chemical agents such as beta-propiolactone, which is effective in the treatment of nonenveloped viruses but has the disadvantage of modifying the treated proteins.
It is also known that certain long treatments using pH modification, below pH 4, or the addition of proteases allows inactivation of some nonenveloped viruses such as parvovirus to a reduced extent. However, these treatments also modify the conformation and structure of the treated proteins.
Consequently, it is known that, to date, the majority of the physicochemical treatment steps capable of being used to obtain viral inactivation of blood products are either highly toxic, or unacceptably affect the conformation of the treated proteins, or are ineffective for treating nonenveloped viruses, in particular parvoviruses.
Parvoviruses are small nonenveloped DNA viruses which infect numerous animal species, including man (Handbook of Parvoviruses, Vol. 1, pp. 1-30, Disinfection, Sterilization and Preservation, Fourth Edition, Seymour S. Block, Ed. Lea and Febiger, Philadelphia-London). They are endemic in nature and cause a wide variety of diseases.
Among these, parvovirus B19 is the only known member of the Parvoviridae family which is pathogenic for man. Likewise, murin parvovirus H1 can also infect man.
Parvovirus B19 infection in a healthy man may be asymptomatic or may induce benign diseases (example: fifth disease in children).
On the other hand, in immunodeficient patients or patients suffering from blood disorders, it may lead to chronic anaemias and to transient aplasias which may be associated with haemolytic anaemias.
Furthermore, Parvovirus B19 infection induces apoptosis of erythroid cells in vitro and in vivo (Yaegashi et al, Journal of Infection, Vol. 39, pp. 68-76 (1999).
Passing through the placenta, it may cause intrauterine death. It exhibits a remarkable tropism for the erythroid lines of human haematopoietic progenitor cells.
A recent epidemiological survey has shown that 50 to 60% of the adult French population and 36% of 1- to 15-year-old children have a positive parvovirus serology.
The process of viral DNA has been demonstrated by genetic amplification (PCR) in a number of batches of purified factor VIII concentrates, regardless of the methods of viral inactivation used.
This has been confirmed by the B19+ serology detection, without clinical sign, in 85% of haemophilic children who have received, since birth, only highly purified factor VIII concentrate (FVIII THPSD) used in France since 1988, free of any contamination with enveloped viruses (HIV, HBV, HCV) (Y. Lauriau et al., ler congrxc3xa8s de la Socixc3xa9´txc3xa9 Francaise de Transfusion [1st conference of the French Transfusion Company] (1994)).
This observation indeed demonstrates that, without new methods of viral inactivation, targeted at the selective elimination of parvoviruses, in particular of parvovirus B19, from blood products, the probability of contamination is high.
Parvoviruses are extremely resistant, even at high temperature. Their haemagglutination properties and their infectivity are not affected by chemical treatments, such as chloroform or various acids, and most resist enzymatic digestions, using RNase, DNase, papain or trypsin.
International Patent Application WO95/00631 describes a method of viral inactivation of blood products comprising the addition to these blood products of products which are photoactivable by UVA radiation and which would become toxic for the viruses present in these blood products. This method comprises a step which makes it possible to isolate these toxic reagents from the blood products so that the latter are not contaminated with these toxic agents.
Among these toxic agents, psoralen may be mentioned in particular.
However, this method has the disadvantage that it cannot be guaranteed that the treated blood products will not be completely free of these photoactivable agents which would be capable of denaturing and/or inactivating the treated blood products and causing toxicity in man or animals when they are reinjected repeatedly, even at a low dose, with the treated blood products.
It is also known that it is possible to sterilize a large number of products by subjecting them to ultraviolet radiation. It is in particular known from the document xe2x80x9cSterilization by Ultraviolet Irradiationxe2x80x9d (chapter 31, IL SHECHMETSTER) that ultraviolet radiation is capable of destroying contaminants such as viruses, mycoplasmas, bacteria and fungi. Such a radiation may be used in particular in media such as gases or liquids.
It is also known from the document by Chin S. et al. (Blood, volume 86, No. 11, December 1995, p. 4331-4336) to treat blood products with type C ultraviolet radiation in the presence or in the absence of antioxidants such as rutin and to obtain the inactivation of nonenveloped viruses, particularly parvoviruses.
In addition, the methods of viral inactivation of the state of the art can affect the integrity and the activity of blood products (in particular the three-dimensional conformation of clotting factors such as factor VIII) and consequently their activities.
Furthermore, the methods of viral inactivation of the state of the art often exhibit difficulties in relation to their validation, because they exhibit problems of reproducibility or of monitoring. Indeed, certain treatment parameters must be modified or cannot be easily maintained, in particular when the degree of humidity has to be monitored if a treatment is carried out with dry heat. Furthermore, it is difficult to control the various steps of the operating procedures.
The present invention aims to obtain a new method and an apparatus for inactivating contaminants present in blood products, which do not exhibit the disadvantages of the state of the art and which are simple, rapid, inexpensive and reproducible.
Another aim of the present invention is to develop a method of viral inactivation which preserves the integrity of blood products, including cells, in particular that of clotting factors such as factor VIII, factor IX, factor VII, factor II, factor X, von Willebrand""s factor, fibronectin, fibrinogen, albunin, immunoglobulin, thrombin, anti-thrombin II, plasminogen LDL, lymphokines, interferons and the like.
A further aim of the present invention is to obtain a method and an apparatus which can be easily validated and which are in accordance with good pharmaceutical manufacturing practices (GMP) and with European standards (CPMP).
A last aim of the present invention is to obtain a method and an apparatus for viral inactivation of blood products which make it possible to inactivate enveloped viruses or nonenveloped viruses, preferably single-stranded, viruses such as parvoviruses, in particular parvoviruses B19 and H1 or HAV. The present invention also airs to obtain the said blood products tree of the said contaminants, in particular of nonenveloped viruses such as parvoviruses, in particular parvoviruses B19 and H1, without the activity of the blood product being affected.
The present invention relates to a new method of inactivating parvoviruses, in particular parvoviruses B19 and H1, present in a blood product, according to which the said blood product is subjected to one or more emission(s) of type C ultraviolet radiation.
xe2x80x9cBlood productxe2x80x9d is understood to mean any blood product or a mix of blood protein compounds, under liquid or solid form, obtained naturally from the human or animal body or by the synthesis route (including genetic engineering) such as whole blood, its cellular compounds (such as erythrocytes, lymphocytes, monocytes and thrombocytes), its derivatives such as serum or plasma and blood protein compounds, namely clotting factors (factor VIII, factor IX, factor VII, factor II, factor X, von Willebrand""s factor and the like), fibrinogen, fibronectin, immunoglobulins, albumin, thrombin, anti-thrombin II, plasminogen, LDL, lymphokines, interferons and the like, including protein compounds obtained by biological engineering, such as recombinant proteins or synthetic peptides.
These products may also be factors produced by certain specific blood cell lines,such as interferons, interleukins, or cell receptors, for these molecules obtained naturally or by the synthetic route, particularly the recombinant peptides or proteins obtained by the recombinant DNA technique. Advantageously, this method also causes inactivation of other contaminating agents such as nonenveloped viruses (HAV), enveloped viruses (HIV, hepatitis B, C, D, E and G viruses and the like), bacterial or fungi, including yeast agents and the like, which may be present in the blood product.
The method according to the invention may also be combined with one or more additional treatment(s) for inactivation of contaminants, particularly viral contaminants, well known to persons skilled in the art, in particular physical or chemical viral inactivation treatments chosen from the group consisting of one or more dry or wet heating step(s), the addition of chemical components, in particular of solvent-detergent or products which become active under ultraviolet radiation, one or more pasteurization step(s), subjection to one or more emissions of particular radiation such as xcex3 radiation or X-rays or a combination of these methods. Among the active products capable of being added to blood products, there may be mentioned in particular agents which protect against free radicals (vitamin C and the like) and betapropiolactone which causes a phenomenon of alkylation of proteins or combined with physical treatment such as nanofiltration by means comprising pores of less than 75, 35 or 15 nm (Asahi, Millipore, Planova, Pall, . . . ). Such products should be used at doses which do not cause a toxicity phenomenon or denaturation of the treated blood products. However, at the irradiation doses used according to the invention, the addition of such products is not necessary in order to cause inactivation of nonenveloped viruses or to ensure protection against free radicals, even for cells treated.
The method of viral inactivation of the invention may be combined with a general method of isolating or separating blood derivatives from whole blood.
This method may comprise one or more filtration, precipitation or chromatographic separation step(s) and the like which make it possible to separate the various components of whole blood from each other.
According to the invention, most of the emission of UVC radiation occurs between about 250 and about 270 nm, preferably at the wavelength of about 254 nm, that is to say the preferred region of absorption of nucleic acids.
Therefore the method of viral inactivation, preferably the method of inactivity present in a blood-product, comprising the use of irradiation dose of ultraviolet radiation received by the blood product lower than 640 joules/m2 (but higher than 2 joules/m2). Preferably the irradiation doses received by the blood products are comprised between about 5 or about 10 joules/m2 and about 500 joules/m2, more preferably between about 200 joules/m2 and about 450 joules/m2, more particularly between about 230 joules/m2 and about 400 joules/m2.
Advantageously, the method according to the invention comprises a step of flowing the blood product past one or more emissions of UVC radiation (type C-ultraviolet radiation), while controlling the flow of the blood product such as the radiation doses of ultraviolet radiation which are received by said blood product, are lower than 640 joules/m2, preferably in the range above described, while the blood product is substantially free of non-envelopped viruses and does not contain quenchers when said blood product is subjected to one or more emissions of type C-ultraviolet radiation.
The inventors have also discovered unexpectedly that it is possible to use said efficient UVC-radiation upon various blood products, while maintaining most of their proteinic activity.
According to the type of blood product treated and to the type of activity bio-assay used, it is possible to test which percentage of activity will be remain after UVC treatment. Said percentage of activity will be advantageously higher than 65 or 70%, preferably higher than 75% and more preferably higher than 80% or 85% or higher than 90% or 95% (the activity of the product not treated will be about 100%).
It is important to note that this percentage can vary a lot according to the blood product treated, but also according to its concentration. The remaining activity is easily characterised for the main blood products such as coagulation factors, but could not be characterised for some specific blood products, such as albumin, immoglobulin, whole blood or cellular compounds.
However, the inventors have observed unexpectedly that the UVC-radiation treatment according to the invention is sufficient to allow an efficient elimination of the viral components presented in said blood products, especially non-envelopped viruses and possibly envelopped viruses.
This method is also easy and safe to be used and does not require the addition of possible toxic compounds, such as quenchers, but could be combined with the addition of compounds such as the one above-mentioned (vitamin C), allowing a protection of the proteinic elements and cellular components present in said blood product against free radicals.
Therefore, the method according to the invention, allows that the radiation doses received by the treated blood product affect essentially the nucleic acid sequences of the viruses, resulting in disrupting and alterating of their nuclear structure.
Unexpectedly, the Inventors have observed that it was also possible to treat blood products not only in thin layers (xe2x80x9cmonolayersxe2x80x9d or so-called laminar layers) or otherwise, that is to say that there are no limiting factors for the volumes treated. This property is particularly advantageous, because by treating blood products which are not in thin layers, it is possible to avoid the disruption phenomena which exist at the solid/liquid surface when the work is carried out in thin layers. In addition, by not working in thin layers, it is possible to treat large quantities of blood products and to avoid problems of heating, shearing and heterogenity of the blood product treated (BAILEY et al., Biochemical Engineering Fundamentals, Vol. II., McGraw-Hill Book Company, 1986).
The wavelength of emission of UVC radiation and the irradiation doses can be adjusted by the person skilled in the art according to the quantity and type of blood products to be treated. However, in order to reduce the phenomenon of denaturation of the blood product, the person skilled in the art may adjust the irradiation doses of the UVC emission wavelength so as to reduce the denaturation and the possible loss of activity of the said blood products. This adjustment will be made so as to be in accordance with the European CPMP standards (CPMP/BWP268/95 and 269/95 rev 2).
It is possible to obtain complete viral inactivation of the non enveloped viruses, especially the parvoviruses present (that is to say that it is no longer possible to identify viruses above the detection threshold) while limiting the irradiation doses received and allowing a reduction in loss of activity of the said product of less than 30-5%, preferably less than 15-5%.
Another aspect of the invention concerns a device or apparatus for the viral inactivation of a blood product, comprising an emitter of type C-ultraviolet array, placed as if to emit the ultraviolet radiation towards the blood product to be treated and placed in a quartz tube or tube made of polymerized material which generally does not absorb in the region of wavelength emitted by type C-ultraviolet radiation and a flow meter controlling the flow rate of the blood product to be treated.
The device or apparatus according to the invention is also not limited to the dose of irradiation used in the method according to the invention and could be used in order to inactivate strongly all the viral elements present in said blood product, even if it may affect the activity of the protein or cellular components comprised therein or softly the cellular components of a blood product to obtain the apoptosis of the cells present in said blood product.
Possibly, the flowmeter, may be integrated directly in the (digital) pump (Watson(copyright) pump).
Said device or apparatus presents the advantages of being simple and easy to be used and could be combined with classical and industrial separation units of blood products. In addition, it does not necessarily need an additional unit for the purification of the blood products in order to remove any addition of possible toxic, chemical components (such as quenchers).
The device or apparatus according to the invention essentially comprises an emitter of type C-ultraviolet arrays, that is to say an emitter whose array length is essentially between about 230 and about 270 nm, preferably at the wave length of the order of 254 nm, which is the maximum region of absorption of ultraviolet array by nucleic acid of the virus to be treated in a blood product.
The device or apparatus according to the invention allows the emission of irradiation doses (to be received by the blood product) of between about 2 joules/m2 and about 2000 joules/m2, preferably lower than 640 joules/m2 (which are advantageously the efficient dose of irradiation in order to obtain the inactivation of non-developed viruses present in said blood product), more preferably irradiation comprised between about 200 and about 500 joules/m2, more preferably between about 240 and about 400 joules/m2.
Therefore, the device or apparatus according to the invention is not limited by the dose of radiation to be received by the blood products and the possible percentage of proteinic inactivation of the blood products to be treated. (However, if the concentration is higher, it is also necessary to increase the irradiation dose (Beer-Lambert Law).
This apparatus also comprises devices which ensure the isolation or the separation of blood derivatives from whole blood.
These devices may comprise means for a precipitation, centrifugation/decantation, filtration, concentration or dialysis of the blood product to be treated which can be adjusted by the person skilled in the art according to the blood products to be separated and treated.
The device or apparatus may also comprise means allowing the addition, to the blood product(s), of an agent which protects against free radicals capable of being generated by the ultraviolet radiation. Such agents may consist of vitamins such as sodium ascorbate, glutathion, or other products (SOD) well known to the person skilled in the art. In addition, the apparatus may also comprise means allowing the addition, to the blood product, of various chemical compounds capable of inactivating certain contaminants present in the blood products to be treated. These compounds may be in particular products which become active under ultraviolet radiation and which are capable of being combined with the method of the invention so as to obtain a synergistic effect on other contaminants present in the said blood product(s).
Another aspect of the present invention is related to a method for treating a mammal patient, including the human, suffering from an hyper-proliferative disorder, such as a leucemie, said method comprising the steps of:
obtaining a blood product from said patient and comprising a cellular component selected from the group consisting of lymphocytes, monocytes or a mixture thereof, in order to obtain their apoptosis,
separating from said blood product specific cells which are not able to be affected by said disorder (for instance erythrocytes, thrombocytes, etc),
flowing the blood product comprising said cells to pass one or more emissions of UVC radiations (type C ultraviolet radiation), while controlling the flow of the blood product such as the radiation doses of the ultraviolet irradiation which are received by said blood product are higher than about 2 joules/m2 and lower than about 50 joules/m2 preferably comprised between about 5 and about 20 joules/m2, and preferably said blood product does not contain quenchers when said blood product is subjected to one or more emissions of said C ultraviolet irradiation and
(possibly) the step of reinjecting said blood product to the patient.
The present invention also relates to the blood product obtained by the method of the invention free of viral contaminants, in particular, free of nonenveloped (single-stranded or double-stranded DNA or RNA) viruses, particularly parvoviruses such as parvoviruses B19 and/or H1, said blood products, in particular blood derivatives such as clotting factors, being characterized by an initial activity of more than 70%, 75%, 80% or 85%, preferably more than 95%.
A final aspect of the present invention relates to the pharmaceutical and/or cosmetic composition (such as a biological adhesive) comprising said blood product(s). The present invention will be described in details in the following non limiting examples with reference to the enclosed figures.